**1. Introduction**

The incidence of many chronic disorders, such as cardiovascular diseases and certain types of cancers, could be attenuated by an improved diet, particularly through increased consumption of diets rich in fruits and vegetables. Such health-promoting properties of edible plants are related mainly to the presence of secondary metabolites, known as phytochemicals [1]. In contrast to traditional pharmaceutical drugs based on single defined substances, herbal preparations have been characterized by a multi-ingredient nature. However, recently this strategy has been introduced in modern medicine with the use of multi-ingredient pharmaceutical cocktails, now prevalent in the treatment of some diseases [2].

The exploitation of olive leaves as a natural resource rich in bioactive compounds would involve the valorization of this byproduct [3]. In addition, olive leaves have a high potential for exploitation in the food industry. Properties of olive leaves are generally attributed to the presence of a range of triterpenes and phenolic compounds such as secoiridoids, lignans, flavonoids, etc. [4].

Fresh olive leaves generally need drying and milling before any purpose. As a preservation method, drying is carried out to remove the water from the leaves to protect the leaves against spoilage and degradation of oleuropein by enzyme action. It also improves extraction efficiency or extractability [5]. Thus, it is considered as the main process in olive leaf treatment. Olive leaves have to be dried for use as an ingredient in dry mixes, extracting phenolic compounds having antioxidant properties, and use in olive leaf tea [6]. In fact, the immediate drying of olive leaves is the most important operation in post-harvest processing in order to avoid quality losses and prevent possible degradation during storage [7], since drying might affect the product quality and is an energy-intensive process [8].

Thus, this work has focused on the determination of phenolic compounds and triterpenoids from fresh and dried olive leaves from El Hor cultivar, cultivated in the center of Tunisia at four sampling dates (January, April, August, and November) using an HPLC-ESI-TOF MS platform. The choice of this olive cultivar was based on our previous findings demonstrating that El Hor olive leaves extracted by supercritical fluid extraction showed the best anticancer activity among other cultivars and extraction techniques [9,10]. Thus, for in-depth study, the aim of this work was to study how the drying process and collecting period of olive leaves affect the phenolic composition of extracts in order to obtain extracts rich in bioactive compounds.

### **2. Results and Discussion**

### *2.1. Identification of Phytochemicals in Olive Leaf Extracts*

Natural antioxidants are mainly secondary metabolites such as phenolic acids, flavonoids, and terpenoids, which are produced by plants for sustaining growth under adverse environment [11]. Polyphenols are among the most widespread class of secondary metabolites in nature, which possess an aromatic ring with one or more hydroxyl substituents.

In the present study, olive leaf extracts were analyzed by HPLC with TOF/MS detection. The characterization process was conducted using the elution order, the interpretation of their mass spectrum provided by the TOF–MS, commercial standards when available, and the data previously reported in the literature. Table 1 includes the compounds, which were identified in olive leaves' SFE extracts, and the information generated by the TOF analyzer—retention time, experimental and calculated *m/z*, molecular formula, and error and milliSigma value. A total of 20 compounds were characterized in SFE extracts by the HPLC-ESI-TOF/MS analytical methods described above. Among them, 17 compounds were from different polar compound classes and polyphenolic families. We classify them into groups such as secoiridoids and related derivatives, simple phenolic compounds, flavonoids (flavonols, flavones, and *O*-methylated flavones), and lignans. In addition, three were characterized as triterpenoids.


**Table 1.** Mass spectral data of the phytochemicals identified in the olive leaf extracts.

\* Identified on the basis of mass spectra. Comparison with the literature, # Identified on the basis of external pure standard.

Figure 1 shows the resulting base peak chromatograms (BPCs) of fresh and dried olive leaf samples collected in January from A1 to A7 according to drying temperature. The main phytochemicals identified are included in Table 1, and their corresponding extracted ion chromatograms (EICs) are shown in Figure 2.

**Figure 1.** Base peak chromatograms (BPCs) of fresh and dried olive leaf samples collected in January from A1 to A7 according to drying temperature. **A1**: Fresh leaves, **A2**: leaves dried at 25 ◦C, **A3**: leaves dried at 40 ◦C, **A4**: leaves dried at 60 ◦C, **A5**: leaves dried at 80 ◦C, **A6**: leaves dried at 100 ◦C, **A7**: leaves dried at 120 ◦C.

**Figure 2.** Extracted ion chromatograms (EICs) of identified compounds in olive leaf samples.

### 2.1.1. Simple Phenolic Compounds

One phenyl alcohol (peak 2 eluted at 8.9 min), aldehyde (peak 6 eluted at 11.7 min), and hydroxycinnamic acid (peak 8 eluted at 14.63 min) were identified in our extracts as hydroxytyrosol, vanillin, and ferulic acid, respectively. The identification of these compounds was confirmed with their corresponding analytical standards.
